&#34;Methods of Reducing Nephrotoxicity in Subjects Administered Nucleoside Phosphonates&#34;

ABSTRACT

A conjugate compound comprising an acyclic nucleoside phosphonate covalently coupled to a lipid for the therapeutic and/or prophylactic treatment of viral infection in an immunodeficient subject is described, along with compositions and methods of using the same. A preferred conugate compound is CMX001, having formula (I) or a pharmaceutically acceptable salt thereof.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 60/914,532, filed Apr. 27, 2007, the disclosure ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention concerns methods of treatment with nucleosidephosphonates, compositions useful in such methods, and the use of suchcompounds.

BACKGROUND OF THE INVENTION

Cidofovir (VISTIDE®) is a nucleoside analog approved by the US FDA forthe treatment of CMV retinitis in patients with AIDS. It is activeagainst all dsDNA viruses that cause human disease. Cidofovir has thestructure:

Cidofovir requires intravenous infusion and is dose-limited by itsnephrotoxicity. Cases of acute renal failure resulting in dialysisand/or contributing to death have occurred with as few as one or twodoses of VISTIDE® Cidofovir. See, e.g., Gilead Letter, Important DrugWarning (September 1996) (available from the US FDA). Hence,prehydration with normal saline and probenecid co-administration arerequired with Cidofovir therapy. See, e.g., S. Lacy, ToxicologicalSciences 44, 97-106 (1998).

U.S. Pat. Nos. 6,716,825; 7,034,014; 7,094,772; and 7,098,197, toHostetler et al. describe lipid conjugates of phosphonate compounds,including cidofovir, for the treatment of disease.

SUMMARY OF THE INVENTION

The present invention provides a conjugate compound comprising anacyclic nucleoside phosphonate covalently coupled to a lipid for thetherapeutic and/or prophylactic treatment of viral infection in animmunodeficient subject.

Preferably the conjugate compound comprises a phosphonate of anantiviral compound of the formula:

or an enantiomer, diastereomer, racemate, stereoisomer, tautomer,rotamer or a mixture thereof, wherein:

R¹ is hydrogen, —CH₃, —CH₂OH, —CH₂F, —CH═CH₂, or —CH₂N₃;

R² is hydrogen; and

B is a purine or pyrimindine;

covalently linked to an alkylglycerol, alkylpropanediol,1-S-alkylthioglycerol, alkoxyalkanol, alkylethanediol,hexadecylpropanediol or octadecylpropanediol;

or a pharmaceutically acceptable salt thereof.

In one embodiment of the invention, the conjugate compound is in theform of an enantiomer, diastereoisomer, racemate or a mixture thereof.

Preferably said acyclic nucleoside phosphonate is selected from thegroup consisting of cidofovir, cyclic cidofovir, tenofovir, andadefovir.

In a preferred embodiment of the invention, said conjugate compound is:

or a pharmaceutically acceptable salt thereof.

A conjugate compound of the present invention may be used for thetherapeutic and/or prophylactic treatment of viral infection in animmunodeficient subject wherein the immunodeficient subject has primaryor acquired immunodeficiency.

In one embodiment of the invention, the immunodeficient subject hasacquired immunodeficiency as a result of immunosuppressive therapy.Cyclosporine for example is an immunosuppressant drug widely used inpost-allogeneic organ transplant to reduce the activity of the patient'simmune system and so the risk of organ rejection. In one embodiment ofthe invention therefore, the immunodeficient subject is a transplantpatient. The immunodeficient subject may be a renal transplant patient,a hepatic transplant patient or a bone marrow transplant patient. In analternative embodiment of the invention, the subject is suffering fromchronic fatigue syndrome.

In one embodiment of the invention, the viral infection to be treated isresistant to treatment with an unconjugated acyclic nucleosidephosphonate, e.g., cidofovir, cyclic cidofovir, tenofovir, and adefovir,etc. Alternatively or additionally, an unconjugated acyclic nucleosidephosphonate exhibits toxic side effects in said immunodeficient subject.

Preferably the immunodeficient subject is infected with at least onedsDNA virus. The dsDNA virus may be selected from any of the groupsconsisting of: human immunodeficiency virus (HIV), influenza, herpessimplex virus (HSV), human herpes virus 6 (HHV-6), cytomegalovirus(CMV), hepatitis B and C virus, Epstein-Barr virus (EBV), varicellazoster virus, variola major and minor, vaccinia, smallpox, cowpox,camelpox, monkeypox, ebola virus, papilloma virus, adenovirus or polyomaviruses including John Cunningham virus (JCV), BK virus and Simianvacuolating virus 40 or Simian virus 40 (SV40).

In one embodiment of the invention, the immunodeficient subject isinfected with a virus or any combination of viruses selected from thegroups consisting of: HCMV, BK virus, HHV-6, Adenovirus and EBV.

In another embodiment of the invention, the immunodeficient subject isinfected with two or more viruses, at least one of which is preferably adsDNA virus, and the viruses exhibit synergistic action. Preferably theviruses are HCMV and BK.

Preferably the conjugate compound is used to treat a dsDNA viralinfection in an immunodeficient subject wherein said subject isresistant to valganciclovir hydrochloride (or ganciclovir) or whereinsaid subject exhibits side effects to valganciclovir hydrochloride (organciclovir). Alternatively or additionally, the conjugate is used totreat cytomegalovirus (CMV) subsequent to treatment with (val)ganciclovir, preferably wherein the CMV infection is emergent. Thepatient may be a bone marrow stem cell transplant patient, especiallywhere there is a risk (real or perceived) for bone marrow toxicity fromganciclovir in the patient.

In a preferred embodiment of the invention, the immunodeficient subjectis a human subject.

Preferably the conjugate compound of the invention is administeredorally, preferably at a dosage of less than 5 mg/Kg, more preferably ata dosage of less than 1 mg/Kg. More preferably said conjugate compoundis administered to said subject at a dosage of 10 or 20, up to 200 or300 or up to 5000 ug/Kg. The lipid conjugates of the invention can beadministered daily, every other day, once a week or once every 2 weeks.

The present invention also provides for the use of a conjugate compoundcomprising an acyclic nucleoside phosphonate covalently coupled to alipid in the manufacture of a medicament for the therapeutic and/orprophylactic treatment of viral infection in an immunodeficient subject.

In another aspect the invention provides a method for the therapeuticand/or prophylactic treatment of viral infection in an immunodeficientsubject, the method comprising administering a conjugate compound to thesubject, said conjugate compound comprising an acyclic nucleosidephosphonate covalently coupled to a lipid.

Preferably said conjugate compound comprises a phosphonate of anantiviral compound of the formula:

or an enantiomer, diastereomer, racemate, stereoisomer, tautomer,rotamer or a mixture thereof, wherein:

R¹ is hydrogen, —CH₃, —CH₂OH, —CH₂F, —CH═CH₂, or —CH₂N₃;

R² is hydrogen; and

B is a purine or pyrimindine;

covalently linked to an alkylglycerol, alkylpropanediol,1-S-alkylthioglycerol, alkoxyalkanol, alkylethanediol,hexadecylpropanediol or octadecylpropanediol;

or a pharmaceutically acceptable salt thereof.

Preferably said compound is in the form of an enantiomer,diastereoisomer, racemate or a mixture thereof. More preferably saidacyclic nucleoside phosphonate is selected from the group consisting ofcidofovir, cyclic cidofovir, tenofovir, and adefovir.

In a preferred embodiment, the conjugate compound is:

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasma concentration curves of CMX001 following a singledose administration; and

FIG. 2, shows plasma concentration curves of Cidofovir following asingle dose of CMX001.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides, among other things, amethod of treating a subject (e.g., a human subject) with an acyclicnucleoside phosphonate, which acyclic nucleoside phosphonate (alsosometimes referred to as an acyclic phosphonate nucleoside herein)induces nephrotoxicity in said subject, the improvement comprising:administering (e.g., oral administering) said acyclic nucleosidephosphonate as a conjugate compound so that said nephrotoxicity isreduced, said conjugate compound comprising said acyclic nucleosidephosphonate covalently coupled to a lipid. In some embodiments, theconjugate compound selected from the group consisting of a phosphonateof an antiviral compound of the formula:

or an enantiomer, diastereomer, racemate, stereoisomer, tautomer,rotamer or a mixture thereof, wherein: R′ is hydrogen, —CH₃, —CH₂OH,—CH₂F, —CH═CH₂, or —CH₂N₃; R² is hydrogen; and B is a purine orpyrimindine covalently linked to an alkylglycerol, alkylpropanediol,1-S-alkylthioglycerol, alkoxyalkanol or alkylethanediol; or apharmaceutically acceptable salt thereof. In some embodiments, thecovalent link is to alkylglycerol, alkylpropanediol,1-S-alkylthioglycerol, alkoxyalkanol, alkylethanediol,octadecylpropanediol, alkylglycerol, hexadecylpropanediol, oroctadecylpropanediol. In some embodiments, the compound is:

or a pharmaceutically acceptable salt thereof. In some embodiments, theacyclic nucleoside phosphonate is selected from the group consisting ofcidofovir, cyclic cidofovir, tenofovir, and adefovir. In someembodiments, the conjugate compound is administered to said subject at adosage of less than 1 mg/Kg; in some embodiments the conjugate compoundis administered to said subject at a dosage of 10 or 20 up to 200 or 300μg/Kg. Also provided is the use of an acyclic nucleoside phosphonate orlipid conjugate thereof as described above for the preparation of amedicament for reducing nephrotoxicity in a subject being treated withan acyclic nucleoside phosphonate according to a method as describedabove.

In some embodiments, the present invention is particularly useful intreating subjects afflicted with at least two different dsDNA whichsynergistically activate one another (e.g., CMV and HIV virus incombination, CMV and BK virus in combination; etc.) See, e.g., LTFeldman et al., PNAS, Aug. 15, 1982, 4952-4956; B. Bielora et al., BoneMarrow Transplant, 2001 September; 28(6): 613-4.

The present invention is explained in greater detail below.

A. Definitions.

“Alkyl” as used herein refers to a monovalent straight or branched chainor cyclic radical of from one to twenty-four carbon atoms, includingmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,n-hexyl, and the like.

“Substituted alkyl” as used herein comprises alkyl groups furtherbearing one or more substituents selected from hydroxy, alkoxy (of alower alkyl group), mercapto (of a lower alkyl group), cycloalkyl,substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl,substituted aryl, heteroaryl, substituted heteroaryl, aryloxy,substituted aryloxy, halogen, trifluoromethyl, cyano, nitro, nitrone,amino, amido, —C(O)H, acyl, oxyacyl, carboxyl, carbamate, sulfonyl,sulfonamide, sulfuryl, and the like.

“Alkenyl” as used herein refers to straight or branched chainhydrocarbyl groups having one or more carbon-carbon double bonds, andhaving in the range of about 2 up to 24 carbon atoms, and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

“Aryl” as used herein refers to aromatic groups having in the range of 6up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

“Heteroaryl” as used herein refers to aromatic groups containing one ormore heteroatoms (e.g., N, O, S, or the like) as part of the ringstructure, and having in the range of 3 up to 14 carbon atoms and“substituted heteroaryl” refers to heteroaryl groups further bearing oneor more substituents as set forth above.

“Bond” or “valence bond” as used herein refers to a linkage betweenatoms consisting of an electron pair.

“Pharmaceutically acceptable salts” as used herein refers to both acidand base addition salts.

“Prodrug” as used herein refers to derivatives of pharmaceuticallyactive compounds that have chemically or metabolically cleavable groupsand become the pharmaceutically active compound by solvolysis or underin vivo physiological conditions.

“Parenteral” as used herein refers to subcutaneous, intravenous,intra-arterial, intramuscular or intravitreal injection, or infusiontechniques.

“Topically” as used herein encompasses administration rectally and byinhalation spray, as well as the more common routes of the skin andmucous membranes of the mouth and nose and in toothpaste.

“Effective amount” as used herein as applied to the phosphonate prodrugsof the invention is an amount that will prevent or reverse the disordersnoted above. Particularly with respect to disorders associated with bonemetabolism, an effective amount is an amount that will prevent,attenuate, or reverse abnormal or excessive bone resorption or the boneresorption that occurs in the aged, particularly post-menopausal femalesor prevent or oppose bone metastasis and visceral metastasis in breastcancer.

“Immunodeficiency” (or “immune deficiency”) as used herein refers to astate in which the ability of the immune system to fight infectiousdisease is compromised or entirely absent. A person who has animmunodeficiency of any kind is said to be immunocompromised. Animmunocompromised person may be particularly vulnerable to opportunisticinfections, in addition to normal infections.

“Treatment” as used herein includes any procedure with a purpose toprevent, pre-empt, treat or cure a disease. Prophylactic treatment mayinclude either primary prophylaxis (to prevent the development of adisease) and/or secondary prophylaxis (whereby the disease has alreadydeveloped and the patient is protected against worsening of thisprocess).

B. Compounds.

Compounds, compositions, formulations, and methods of treating subjectsthat can be used to carry out the present invention include but are notlimited to those described in U.S. Pat. Nos. 6,716,825; 7,034,014;7,094,772; and 7,098,197, the disclosures of which are incorporated byreference herein in their entirety.

In some embodiments the phosphonate compounds of the invention have thestructure:

wherein:

R₁ and R₁′ are independently —H, optionally substituted —O(C₁-C₂₄)alkyl,—O(C₁-C₂₄)alkenyl, —O(C₁-C₂₄)acyl, —S(C₁-C₂₄)alkyl, —S(C₁-C₂₄)alkenyl,or —S(C₁-C₂₄)acyl, wherein at least one of R₁ and R₁′ are not —H, andwherein said alkenyl or acyl moieties optionally have 1 to 6 doublebonds,

R₂ and R₂′ are independently —H, optionally substituted —O(C₁-C₇)alkyl,—O(C₁-C₇)alkenyl, —S(C₁-C₇)alkyl, —S(C₁-C₇)alkenyl, —O(C₁-C₇)acyl,—S(C₁-C₇)acyl, —N(C₁-C₇)acyl, —NH(C₁-C₇)alkyl, —N((C₁-C₇)alkyl)₂, oxo,halogen, —NH₂, —OH, or —SH;

R₃ is a pharmaceutically active phosphonate, bisphosphonate or aphosphonate derivative of a pharmacologically active compound, linked toa functional group on optional linker L or to an available oxygen atomon C_(α);

X, when present, is:

L is a valence bond or a bifunctional linking molecule of the formula-J-(CR₂)_(t)-G-, wherein t is an integer from 1 to 24, J and G areindependently —O—, —S—, —C(O)O—, or —NH—, and R is —H, substituted orunsubstituted alkyl, or alkenyl;

m is an integer from 0 to 6; and

n is 0 or 1.

In some embodiments, m=0, 1 or 2. In these embodiments, R₂ and R₂′ arepreferably H, and the prodrugs are then ethanediol, propanediol orbutanediol derivatives of a therapeutic phosphonate. A preferredethanediol phosphonate species has the structure:

wherein R₁, R₁′, R₃, L, and n are as defined above.

One propanediol species has the structure:

wherein m=1 and R₁, R₁′, R₃, L and n are as defined above in the generalformula.

A glycerol species has the structure:

wherein m=1, R₂═H, R₂′═OH, and R₂ and R₂′ on C^(α) are both —H. Glycerolis an optically active molecule. Using the stereospecific numberingconvention for glycerol, the sn-3 position is the position which isphosphorylated by glycerol kinase. In compounds of the invention havinga glycerol residue, the -(L)_(n)-₃ moiety may be joined at either thesn-3 or sn-1 position of glycerol.

In all species of the pharmacologically active agents of the invention,R₁ is preferably an alkoxy group having the formula —O—(CH₂)_(t)—CH₃,wherein t is 0-24. More preferably t is 11-19. Most preferably t is 15or 17.

Some examples of antiviral phosphonates derived by substituting—CH₂—PO₃H₂ for the 5′-hydroxyl are: AZT phosphonate, d4T phosphonate,ddC phosphonate, Adefovir, ganciclovir phosphonate, acyclovirphosphonate, ganciclovir cycloic phosphonate, and3′-thia-2′,3′-dideoxycytidine-5′-phosphonic acid

Other examples of antiviral nucleotide phosphonates contemplated for usein the practice of the invention are derived similarly from antiviralnucleosides including ddA, ddI, ddG, L-FMAU, DXG, DAPD, L-dA, L-dI,L-(d)T, L-dC, L-dG, FTC, penciclovir, and the like.

Additionally, antiviral phosphonates such as cidofovir, cycliccidofovir, adefovir, tenofovir, and the like, may be used as an R₃ groupin accordance with the present invention.

Certain compounds of the invention possess one or more chiral centers,e.g. in the sugar moieties, and may thus exist in optically activeforms. Likewise, when the compounds contain an alkenyl group or anunsaturated alkyl or acyl moiety there exists the possibility of cis-and trans-isomeric forms of the compounds. Additional asymmetric carbonatoms can be present in a substituent group such as an alkyl group. TheR- and S-isomers and mixtures thereof, including racemic mixtures aswell as mixtures of cis- and trans-isomers are contemplated by thisinvention. All such isomers as well as mixtures thereof are intended tobe included in the invention. If a particular stereoisomer is desired,it can be prepared by methods well known in the art by usingstereospecific reactions with starting materials that contain theasymmetric centers and are already resolved or, alternatively, bymethods that lead to mixtures of the stereoisomers and resolution byknown methods.

Nucleosides useful for treating viral infections may also be convertedto their corresponding 5′-phosphonates for use as an R₃ group. Suchphosphonate analogs typically contain either a phosphonate (—PO₃H₂) or amethylene phosphonate (—CH₂—PO₃H₂) group substituted for the 5′-hydroxylof an antiviral nucleoside. Some examples of antiviral phosphonatesderived by substituting —PO₃H₂ for the 5′-hydroxyl are:

Many phosphonate compounds exist that can be derivatized according tothe invention to improve their pharmacologic activity, or to increasetheir oral absorption, such as, for example, the compounds disclosed inthe following patents, each of which are hereby incorporated byreference in their entirety: U.S. Pat. No. 5,043,437 (Phosphonates ofazidodideoxynucleosides), U.S. Pat. No. 5,047,533 (Acyclic purinephosphonate nucleotide analogs), U.S. Pat. No. 5,142,051(N-Phosphonylmethoxyalkyl derivatives of pyrimidine and purine bases),U.S. Pat. No. 5,247,085 (Antiviral purine compounds), U.S. Pat. No.5,395,826 (Guanidinealky 1-1,1-bisphosphonic acid derivatives), U.S.Pat. No. 5,656,745 (Nucleotide analogs), U.S. Pat. No. 5,672,697(Nucleoside-5′-methylene phosphonates), U.S. Pat. No. 5,717,095(Nucleotide analogs), U.S. Pat. No. 5,760,013 (Thymidylate analogs),U.S. Pat. No. 5,798,340 (Nucleotide analogs), U.S. Pat. No. 5,840,716(Phosphonate nucleotide compounds), U.S. Pat. No. 5,856,314(Thio-substituted, nitrogen-containing, heterocyclic phosphonatecompounds), U.S. Pat. No. 5,885,973 (olpadronate), U.S. Pat. No.5,886,179 (Nucleotide analogs), U.S. Pat. No. 5,877,166(Enantiomericaily pure 2-aminopurine phosphonate nucleotide analogs),U.S. Pat. No. 5,922,695 (Antiviral phosphonomethoxy nucleotide analogs),U.S. Pat. No. 5,922,696 (Ethylenic and allenic phosphonate derivativesof purines), U.S. Pat. No. 5,977,089 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,043,230 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,069,249 (Antiviral phosphonomethoxynucleotide analogs); Belgium Patent No. 672205 (Clodronate); EuropeanPatent No. 753523 (Amino-substituted bisphosphonic acids); EuropeanPatent Application 186405 (geminal diphosphonates); and the like.

Phosphonate analogs, comprising therapeutically effective phosphonates(or phosphonate derivatives of therapeutically effective compounds)covalently linked by a hydroxyl group to a 1-O-alkyglycerol,3-O-alkylglycerol, 1-S-alkylthioglycerol, or alkoxy-alkanol, may beabsorbed more efficiently in the gastrointestinal tract than are theparent compounds. An orally administered dose of the analog is taken upintact from the gastrointestinal tract of a mammal and the active drugis released in vivo by the action of endogenous enzymes. Phosphonateanalogs of the invention may also have a higher degree of bioactivitythan the corresponding underivatized compounds.

The compounds of the present invention are an improvement overalkylglycerol phosphate prodrugs described in the prior art because thephosphonate-containing moiety is linked directly to the alkyl-glycerolor the alkoxy-alkanol moiety and because the presence of the phosphonatebond prevents enzymatic conversion to the free drug. Other linkersbetween these groups can be present in the improved analogs. Forexample, bifunctional linkers having the formula —O—(CH₂)_(n)—C(O)O—wherein n is 1 to 24, can connect the phosphonate to the hydroxyl groupof the alkoxy-alkanol or alkylglycerol moiety.

The foregoing allows the phosphonate of the invention to achieve ahigher degree of oral absorption. Furthermore, cellular enzymes, but notplasma or digestive tract enzymes, will convert the conjugate to a freephosphonate. A further advantage of the alkoxy-alkanol phosphonates isthat the tendency of co-administered food to reduce or abolishphosphonate absorption is greatly reduced or eliminated, resulting inhigher plasma levels and better compliance by patients.

Compounds (or “prodrugs”) useful in the invention can be prepared in avariety of ways, as generally depicted in Schemes I-VI of U.S. Pat. No.6,716,825. The general phosphonate esterification methods describedbelow are provided for illustrative purposes only and are not to beconstrued as limiting this invention in any manner. Indeed, severalmethods have been developed for direct condensation of phosphonic acidswith alcohols (see, for example, R. C. Larock, Comprehensive OrganicTransformations, VCH, New York, 1989, p. 966 and references citedtherein). Isolation and purification of the compounds and intermediatesdescribed in the examples can be effected, if desired, by any suitableseparation or purification procedure such as, for example, filtration,extraction, crystallization, flash column chromatography, thin-layerchromatography, distillation or a combination of these procedures.Specific illustrations of suitable separation and isolation proceduresare in the examples below. Other equivalent separation and isolationprocedures can of course, also be used.

Scheme I of U.S. Pat. No. 6,716,825. outlines a synthesis ofbisphosphonate prodrugs that contain a primary amino group, such aspamidronate or alendronate. Example 1 therein provides conditions for asynthesis of 1-O-hexadecyloxypropyl-alendronate (HDP-alendronate) or1-O-hexadecyloxypropyl-pamidronate (HDP-pamidronate). In this process, amixture oT dimethyl 4-phthalimidobutanoyl phosphonate (1b, prepared asdescribed in U.S. Pat. No. 5,039,819)) and hexadecyloxypropyl methylphosphite (2) in pyridine solution is treated with triethylamine toyield bisphosphonate tetraester 3b which is purified by silica gelchromatography. Intermediate 2 is obtained by transesterification ofdiphenyl phosphite as described in Kers, A., Kers, I., Stawinski, J.,Sobkowski, M., Kraszewski, A. Synthesis, April 1995, 427 430. Thus,diphenyl phosphite in pyridine solution is first treated withhexadecyloxypropan-1-ol, then with methanol to provide compound 2.

An important aspect of the process is that other long chain alcohols maybe used in place of hexadecyloxypropan-1-ol to generate the variouscompounds of this invention. Treatment of intermediate 3b withbromotrimethylsilane in acetonitnle cleaves the methyl estersselectively to yield monoester 4b. Treatment of 4b with hydrazine in amixed solvent system (20% melhanol/80% 1,4-dioxane) results in removalof the phthalimido protecting group as shown. The desired alendronateprodrug is collected by filtration and converted to the triammonium saltby treatment with methanolic ammonia.

Scheme II of U.S. Pat. No. 6,716,825. illustrates a synthesis of analogsof bisphosphonates lacking a primary amino group, hi this case theprocess steps are similar to those of Scheme 1 except that protectionwith a phthalimido group and subsequent deprotection by hydrazinolysisare unnecessary.

Bisphosphonates having 1-amino groups, such as amino-olpadronate, maybeconverted to analogs according to the invention prodrugs using aslightly modified process shown in Scheme III of U.S. Pat. No.6,716,825. Treatment of a mixture of compound 2 and3-(dimethylamino)propionitrile with dry HCl followed by addition ofdimethyl phosphite affords tetraester 3 which, after demethylation withbromotrimethylsilane, yields hexadecyloxypropyl-amino-olpadronate.

Scheme IV of U.S. Pat. No. 6,716,825 illustrates synthesis of abisphosphonate analog where the lipid group is attached to a primaryamino group of the parent compound rather than as a phosphonate ester.

Scheme V of U.S. Pat. No. 6,716,825. illustrates a general synthesis ofalkylglycerol or alkylpropanediol analogs of cidofovir, cycliccidofovir, and other phosphonates. Treatment of 2,3-isopropylideneglycerol, 1, with NaH in dimethylformamide followed by reaction with analkyl methanesulfonate yields the alkyl ether, 2. Removal of theisopropylidene group by treatment with acetic acid followed by reactionwith trityl chloride in pyridine yields the intermediate 3. Alkylationof intermediate 3 with an alkyl halide results in compound 4. Removal ofthe trityl group with 80% aqueous acetic acid affords the O,O-dialkylglycerol, 5. Bromination of compound 5 followed by reaction with thesodium salt of cyclic cidofovir or other phosphonate-containingnucleotide yields the desired phosphonate adduct, 7. Ring-opening of thecyclic adduct is accomplished by reaction with aqueous sodium hydroxide.The preferred propanediol species may be synthesized by substituting1-O-alkylpropane-3-ol for compound 5 in Scheme V. The tenofovir andadefovir analogs may be synthesized by substituting these nucleotidephosphonates for cCDV in reaction (f) of Scheme V. Similarly, othernucleotide phosphonates of the invention may be formed in this manner.

Scheme VI of U.S. Pat. No. 6,716,825. illustrates a general method forthe synthesis of nucleotide phosphonates of the invention using1-O-hexadecyloxypropyl-adefovir as the example. The nucleotidephosphonate (5 mmol) is suspended in dry pyridine and an alkoxyalkanolor alkylglycerol derivative (6 mmol) and 1,3-dicyclohexylcarbodiimde(DCC, 10 mmol) are added. The mixture is heated to reflux and stirredvigorously until the condensation reaction is complete as monitored bythin-layer chromatography. The mixture is then cooled and filtered. Thefiltrate is concentrated under reduced pressure and the residuesadsorbed on silica gel and purified by flash column chromatography(elution with approx. 9:1 dichloromethane/methanol) to yield thecorresponding phosphonate monoester.

C. Compositions.

Compounds of the invention can be administered orally in the form oftablets, capsules, solutions, emulsions or suspensions, inhaled liquidor solid particles, microencapsulated particles, as a spray, through theskin by an appliance such as a transdermal patch, or rectally, forexample, in the form of suppositories. The lipophilic prodrugderivatives of the invention are particularly well suited fortransdermal absorption administration and delivery systems and may alsobe used in toothpaste. Administration can also take place parenterallyin the form of injectable solutions.

The compositions may be prepared in conventional forms, for example,capsules, tablets, aerosols, solutions, suspensions, or together withcarriers for topical applications. Pharmaceutical formulationscontaining compounds of this invention can be prepared by conventionaltechniques, e.g., as described in Remington's Pharmaceutical Sciences,1985.

The pharmaceutical carrier or diluent employed may be a conventionalsolid or liquid carrier. Examples of solid carriers are lactose,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,stearic acid, or lower alkyl ethers of cellulose. Examples of liquidcarriers are syrup, peanut oil, olive oil, phospholipids, fatty acids,fatty acid amines, polyoxyethylene or water. The carrier or diluent mayinclude any sustained release material known in the art, such asglyceryl monostearate or distearate, alone or mixed with a wax.

If a solid carrier is used for oral administration, the preparation maybe tableted or placed in a hard gelatin capsule in powder or pelletform. The amount of solid carrier will vary widely, but will usually befrom about 25 mg to about 1 gm. If a liquid carrier is used, thepreparation may be in the form of a syrup, emulsion, soft gelatincapsule, or sterile injectable liquid such as an aqueous or non-aqueousliquid suspension or solution.

Tablets are prepared by mixing the active ingredient (that is, one ormore compounds of the invention), with pharmaceutically inert, inorganicor organic carrier, diluents, and/or excipients. Examples of suchexcipients which can be used for tablets are lactose, maize starch orderivatives thereof, talc, stearic acid or salts thereof. Examples ofsuitable excipients for gelatin capsules are vegetable oils, waxes,fats, semisolid, and liquid polyols. The bisphosphonate prodrugs canalso be made in microencapsulated form.

For nasal administration, the preparation may contain a compound of theinvention dissolved or suspended in a liquid carrier, in particular, anaqueous carrier, for aerosol application. The carrier may containsolubilizing agents such as propylene glycol, surfactants, absorptionenhancers such as lecithin or cyclodextrin, or preservatives.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or non-aqueousliquids, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use.

Suitable excipients for the preparation of solutions and syrups arewater, polyols, sucrose, invert sugar, glucose, and the like. Suitableexcipients for the preparation of injectable solutions are water,alcohols, polyols, glycerol, vegetable oils, and the like.

The pharmaceutical products can additionally contain any of a variety ofadded components, such as, for example, preservatives, solubilizers,stabilizers, wetting agents, emulsifiers, sweeteners, colorants,flavorings, buffers, coating agents, antioxidants, diluents, and thelike.

Optionally, the pharmaceutical compositions of the invention maycomprise a compound according to the general formula combined with oneor more compounds exhibiting a different activity, for example, anantibiotic or other pharmacologically active material. Such combinationsare within the scope of the invention.

D. Subjects and Methods.

A number of rare diseases feature a heightened susceptibility toinfections from childhood onward. Many of these disorders are hereditaryand are autosomal recessive or X-linked. There are over 80 recognisedprimary immunodeficiency syndromes; they are generally grouped by thepart of the immune system that is malfunctioning, such as lymphocytes orgranulocytes. The treatment of primary immunodeficiencies depends on thenature of the defect, and may involve antibody infusions, long-termantibiotics and (in certain cases) stem cell transplantation.

Immune deficiency may also be the result of particular externalprocesses or diseases; the resultant state is called “secondary” or“acquired” immunodeficiency. Common causes for secondaryimmunodeficiency are malnutrition, aging and particular medications(e.g. chemotherapy, disease-modifying antirheumatic drugs,immunosuppressive drugs after organ transplants, glucocorticoids).

Many specific diseases directly or indirectly impair the immune system.This include many types of cancer, particularly those of the bone marrowand blood cells (leukemia, lymphoma, multiple myeloma), and certainchronic infections. Immunodeficiency is also the hallmark of acquiredimmunodeficiency syndrome (AIDS), caused by the human immunodeficiencyvirus (HIV). HIV directly attacks the immune system.

Human cytomegalovirus (HCMV) is a member of the herpes virus family.These dsDNA viruses typically cause mild or subclincal disease, but cancause severe systemic or localised disease in immunocomprisedindividuals. All herpes viruses share a characteristic ability to remainlatent within the body over long periods. Although primary CMV infectionin an immunocompromised patient can cause serious disease, the morecommon problem is the reactivation of the latent virus.

Immunocompromised patients include organ transplant recipients, patientsundergoing hemodialysis, patients with cancer, patients receivingimmunosuppressive drugs, and HIV-infected patients. Exposure ofimmunosuppressed patients to outside sources of CMV should be minimized.Whenever possible, patients without CMV infection should be given organsand/or blood products that are free of the virus.

Patients without CMV infection who are given organ transplants fromCMV-infected donors should be given prophylactic treatment withvalganciclovir (ideally) or ganciclovir and require regular serologicalmonitoring to detect a rising CMV titre, which should be treated earlyto prevent a potentially life-threatening infection becomingestablished.

However, despite prophylaxis, often continued for 100 days, diseaseprevalence at six months is estimated to be from 12 to 22 percent. Safeand efficacious prophylaxis of CMV infection in transplant patients isnot possible with current treatments.

CMX001 has a decided advantage over current treatments for prophylaxisof CMV infection in transplant recipients and cancer patients receivingmyelosuppressive chemotherapy or radiation therapy, based on thedemonstrated lack of nephrotoxicity at potentially effectiveconcentrations. Large patient populations with needs that couldpreviously not be met with existing therapies can now be treated. Thesurprisingly low levels of nephrotoxicity associated with the compoundsof the present invention means that the application of these compoundsin treatment of immunocompromised individuals is now possible.

Furthermore, although anti-viral therapies have advanced substantiallyin recent years, resistance to current agents and significant drug sideeffects remain an issue for many patients. The conjugate compounds ofthe present invention demonstrate high oral bioavailability at lowerdoses than conventional drugs and this has important implications fordisease resistance.

More generally, this invention provides methods of treating mammaliandisorders related to bone metabolism, viral infections, inappropriatecell proliferation, and the like. The methods particularly compriseadministering to a human or other mammal in need thereof atherapeutically effective amount of the prodrugs of this invention.Indications appropriate to such treatment include senile,post-menopausal or steroid-induced osteoporosis, Paget's disease,metastatic bone cancers, hyperparathyroidism, rheumatoid arthritis,algodystrophy, stemo-costoclavicular hyperostosis, Gaucher's disease,Engleman's disease, certain non-skeletal disorders and periodontaldisease, human immunodeficiency virus (HIV), influenza, herpes simplexvirus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis Bvirus, Epstein-Barr virus (EBV), varicella zoster virus, lymphomas,hematological disorders such as leukemia, and the like.

In accordance with yet another aspect of the invention, there areprovided methods for treating disorders caused by viral infections.Indications appropriate to such treatment include susceptible virusessuch as human immunodeficiency virus (HIV), influenza, herpes simplexvirus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis Band C virus, Epstein-Barr virus (EBV), varicella zoster virus, anddiseases caused by orthopox viruses (e.g., variola major and minor,vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebolavirus, papilloma virus, and the like.

The prodrugs of the invention can be administered orally, parenterally,topically, rectally, and through other routes, with appropriate dosageunits, as desired.

With respect to disorders associated with viral infections, the“effective amount” is determined with reference to the recommendeddosages of the antiviral compound. The selected dosage will varydepending on the activity of the selected compound, the route ofadministration, the severity of the condition being treated, and thecondition and prior medical history of the patient being treated.However, it is within the skill of the art to start doses of thecompound(s) at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. If desired, the effective daily dose may bedivided into multiple doses for purposes of administration, for example,two to four doses per day. It will be understood, however, that thespecific dose level for any particular patient will depend on a varietyof factors, including the body weight, general health, diet, time, androute of administration and combination with other drugs, and theseverity of the disease being treated.

Generally, the compounds of the present invention are dispensed in unitdosage form comprising 1% to 100% of active ingredient. The range oftherapeutic dosage is from about 0.01 to about 1,000 mg/kg/day with fromabout 0.10 mg/kg/day to 100 mg/kg/day being preferred, when administeredto patients, e.g., humans, as a drug. Actual dosage levels of activeingredients in the pharmaceutical compositions of this invention may bevaried so as to administer an amount of the active compound(s) that iseffective to achieve the desired therapeutic response for a particularpatient.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1 Preclinical Studies of CMX001

As summarized in Tables 1-2 below, pre-clinical studies of CMX001indicate that it is essentially completely protective against lethalOrthopoxivirus infections in mice and rabbits. The effective dose inthese animal models ranges from 1-2 mg/kg daily for 5 days in low titerinoculums, while late stage requires 20-30 mg/kg as a single dose.

TABLE 1 CMX001 has Enhanced In Vitro Potency Against dsDNA Viruses. CellCidofovir CMX001 Enhanced Virus Line EC50 (μM) EC50 (μM) ActivityVariola major Vero 76 27.3 0.1 271 Vaccinia Virus HFF 46 0.8 57HCMV(AD169) MRC-5 0.38 0.0009 422 BK Virus WI-38 115.1 0.13 885 HSV-1MRC-5 15 0.06 250 HHV-6 HSB-2 0.2 0.004 50 Adenovirus HFF 1.3 0.02 65HPV 18 HeLa 516 0.42 1229 HPV 11 A431 716 17 42 EBV Dardi >170 0.04>4250

TABLE 2 CMX001 is protective against lethal orthopoxivirus infections inmice and rabbits. Viral Inoculum 100% Protective (PFU) Dose of CMX001*Mice Infected with Ectromelia 1.2 1 mg/kg/day 27 4 mg/kg/day 270 4mg/kg/day 9200 8 mg/kg/day Rabbits Infected with Rabbitpox 100 2mg/kg/day 500 10 mg/kg/day  1000 20 mg/kg/day  *Dose was orallyadministered for five consecutive days

In addition, over twenty-one toxicology studies have been conducted inmice, rats, rabbits and monkeys with CMX001 being delivered by the oralroute. In none of these studies (as opposed to the delivery ofefficacious doses of cidofovir by i.v.), has there been any indicationof nephrotoxicity (see, e.g., Example 2 below).

Example 2 Clinical Studies

An initial study was conducted to evaluate the safety andpharmacokinetics of CMX001 in healthy volunteers. The study consisted ofa single dose arm (SD) and a multiple dose arm (MD). In the single dosearm 7 cohorts of 6 subjects were treated (4 subjects received activedrug and 2 placebo). Enrollment was staggered as 2 subjects (one active,one placebo) followed by 4 subjects (Groups A and B). The estimatedsingle doses for the two highest doses treated for a 75 kg subject were40 mg (0.6 mg/kg cohort 6) and 70 mg (1 mg/kg cohort 7). In the multipledose arm, cohort 6MD received 0.1 mg/kg on Day 0, 6 and 12; Cohort 7MDreceived 0.2 mg/kg on Day 0, 6 and 12. Levels of cidofovir, CMX001 andCMX064 (major metabolite) were measured in blood and urine of subjectsduring the course of the study. Gastrointestinal (GI) monitoring of thesubjects included (a) monitoring for clinical signs of GI adverseevents, (b) monitoring for clinical symptoms using a visual AnalogScale, (c) monitoring for appetite loss/anorexia, nausea, vomiting,diarrhea, constipation and intestinal gas/bloating, (d) laboratory testsfor fecal occult blood; serum electrolytes, urine specific gravity,BUN/creatinine ratio; serum albumin, and lipids, and (e) diagnosticstudies (the Wireless capsule endoscopy (PillCam®, Given Imaging)).

Upon the completion of the study of cohort 6 (600 μg/kg) (while stillblinded) it was observed as follows:

-   -   No post-dose clinically significant gastrointestinal capsule        endoscopy findings attributable to drug.    -   No drug associated clinically significant changes to clinical        laboratory values, including those indicative of kidney        dysfunction.    -   No serious adverse events (SAEs), no significant adverse events        (AEs) (i.e. ≦Grade 2), no AEs directly attributable to drug.        Plasma concentration curves of CMX001 following a single dose        administration are shown in FIG. 1, and plasma concentration        curves of Cidofovir following a single dose of CMX001 are shown        in FIG. 2.

Table 3 illustrates the PK comparison of CMX 001 with CMX 021 and CMX064 for mouse, rabbit and human.

TABLE 3 CMX001 CMX021 CMX064 Dose Cmax AUCO→ Cmax AUCO→ Cmax AUCO→Species (mg/kg) (ng/mL) (ng * h/mL) (ng/mL) (ng * h/mL) (ng/mL) (ng *h/mL) Mouse- 2 7.9-18.0 83.14-102.4 BQL-5.44 ND-50.1 — — Rabbit Human0.025 2.36 18.51 BQL ND 1.69 11.83 0.050 5.63 36.32 1.51 33.28 4.6338.95 0.100 10.62 133.47 3.44 125.14 2.85 34.02 0.200 24.48 225.49 5.41189.92 4.55 39.73 0.400 68.13 526.37 10.44 444.76 23.03 202.99 0.600114.73 728.8 12.19 519.0 24.86 187.0 Calculated based on mouse doses of2 and 10 mg/kg, rabbit doses of 5 and 10 mg/kg ND Not Determined, BQLBelow Quantitation Limit

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-28. (canceled)
 29. A method for treating, preempting or preventingviral infection in an immunodeficient subject comprising administeringto said subject a conjugate compound comprising an acyclic nucleosidephosphonate covalently coupled to a lipid, wherein the conjugatecompound is selected from:

and pharmaceutically acceptable salts thereof; further wherein theimmunodeficient subject is infected with at least one double strandedDNA (dsDNA) virus; further wherein the conjugate compound isadministered to the subject at a dosage of less than 5 mg/kg; andfurther wherein the subject is a human. 30-33. (canceled)
 34. The methodof claim 29, wherein said immunodeficient subject has primary oracquired immunodeficiency.
 35. The method of claim 29, wherein saidimmunodeficient subject has acquired immunodeficiency as a result ofimmunosuppressive therapy.
 36. The method of claim 35, wherein saidimmunodeficient subject has acquired immunodeficiency as a result ofcyclosporine treatment.
 37. The method of claim 29, wherein saidimmunodeficient subject is a transplant patient.
 38. The method of claim29, wherein said immunodeficient subject is a renal transplant patient,a hepatic transplant patient or a bone marrow transplant patient. 39.The method of claim 29, wherein said immunodeficient subject is a humansubject.
 40. The method of claim 29, wherein said immunodeficientsubject is suffering from chronic fatigue syndrome.
 41. The method ofclaim 29, wherein the viral infection is resistant to treatment with theunconjugated acyclic nucleoside phosphonate.
 42. The method of claim 29,wherein the unconjugated acyclic nucleoside phosphonate exhibits toxicside effects in said immunodeficient subject.
 43. (canceled)
 44. Themethod of claim 29, wherein said dsDNA virus is selected from the groupconsisting of: human immunodeficiency virus (HIV), herpes simplex virus(HSV), human herpes virus 6 (HHV-6), human cytomegalovirus (HCMV),hepatitis B virus, hepatitis C virus, Epstein-Barr virus (EBV),varicella zoster virus, variola major and minor, vaccinia, smallpox,cowpox, camelpox, monkeypox, ebola virus, papilloma virus, adenovirus,polyoma virus, JC virus, BK virus, SV40 and a combination thereof. 45.The method of claim 44, wherein said immunodeficient subject is infectedwith a virus or any combination of viruses selected from the groupconsisting of: HCMV, BK virus, HHV-6, adenovirus hepatitis B virus andEBV.
 46. The method of claim 29, wherein said immunodeficient subject isinfected with two or more viruses and said two or more viruses exhibitsynergistic action.
 47. The method of claim 44, wherein said dsDNA virusis selected from the group consisting of: EBV, HCMV, JC virus and BKvirus.
 48. A method of treating a dsDNA viral infection in animmunodeficient subject wherein said subject is resistant tovalganciclovir hydrochloride or ganciclovir or wherein said subjectexhibits side effects to valganciclovir hydrochloride or ganciclovircomprising administering to the subject a conjugate compound selectedfrom:

and pharmaceutically acceptable salts thereof; further wherein theconjugate compound is administered to the subject at a dosage of lessthan 5 mg/kg; and further wherein the subject is a human.
 49. The methodof claim 48, wherein said conjugate compound is administered to thesubject to treat human cytomegalovirus (HCMV) after the subject isadministered valganciclovir hydrochloride or ganciclovir.
 50. (canceled)51. (canceled)
 52. The method of claim 29, wherein said conjugatecompound is administered to said subject at a dosage of about 20 toabout 5000 μg/Kg.
 53. (canceled)
 54. The method of claim 29, whereinsaid immunodeficient subject is infected with human immunodeficiencyvirus.
 55. (canceled)
 56. (canceled)
 57. A method of treating a viralinfection in a subject, comprising: (a) identifying a subject having aviral infection and exhibiting toxic side effects from treatment with anunconjugated acyclic nucleoside phosphonate; and (b) administering tothe subject a conjugate compound selected from:

and pharmaceutically acceptable salts thereof; wherein the subject isinfected with at least one double stranded DNA (dsDNA) virus; andfurther wherein the conjugate compound is administered to the subject ata dosage of less than 5 mg/kg; and further wherein the subject is ahuman. 58-60. (canceled)
 61. The method of claim 29, wherein saidconjugate compound is administered daily.
 62. The method of claim 29,wherein said conjugate compound is administered every other day.
 63. Themethod of claim 29, wherein said conjugate compound is administered oncea week.
 64. The method of claim 29, wherein said conjugate compound isadministered once every two weeks.
 65. The method of claim 29, whereinsaid conjugate compound is administered to said subject at a dosage of1-2 mg/kg daily.
 66. The method of claim 29, wherein said conjugatecompound is administered to said subject at a dosage of less than 1mg/kg.
 67. The method of claim 29, wherein said conjugate compound isadministered to said subject at a dosage of 0.6 mg/kg.
 68. The method ofclaim 29, wherein said conjugate compound is administered to saidsubject at a dosage of 0.1 mg/kg every 6 days.
 69. The method of claim29, wherein said conjugate compound is administered to said subject at adosage of 0.2 mg/kg every 6 days.
 70. The method of claim 29, whereinsaid conjugate compound is

or a pharmaceutically acceptable salt thereof.
 71. The method of claim29, wherein said conjugate compound is

or a pharmaceutically acceptable salt thereof.
 72. The method of claim29, wherein said immunodeficient subject is also infected withinfluenza.
 73. The method of claim 46, wherein said two or more virusesare HCMV and BK virus.
 74. The method of claim 46, wherein said two ormore viruses are HCMV and HIV.